Effective cancer prevention depends on distinguishing between indolent and progressive pre-malignant neoplasms, so that the cost and risks of interventions can be focused on patients likely to progress to cancer, while patients at low risk of progression can be reassured and removed from frequent surveillance. Because neoplastic progression is a process of clonal evolution, we are developing a novel class of biomarkers that directly measure the evolutionary process. We have previously shown that measures of clonal diversity in Barrett's esophagus predict progression to esophageal adenocarcinoma. However, those methods depended on detecting distinct clones by taking multiple, spatially separated biopsies from each neoplasm. This is not feasible for many neoplasms. In order to generalize the use of genetic diversity as a biomarker of progression to other neoplasms, we propose to develop a genetic fingerprinting technique for measuring genetic heterogeneity at the single cell, or single crypt, level. We will flow sort single cells into a 96 well plate and use fluorescent inter-simple sequence repeat PCR (FISSR-PCR) to detect insertions, deletions and translocations in each cell's genome. If single cell assays prove unreliable, we can isolate single whole epithelial crypts into separate wells to characterize genetic diversity at the crypt level. We will test this assay on Barrett's esophagus biopsies from 19 genetically mapped esophagectomy specimens to determine if p53 wildtype epithelium is less genetically diverse than epithelium with p53 loss of heterozygosity, which in turn is less diverse than aneuploid or tetraploid epithelium. This will determine if the molecular events that characterize Barrett's neoplastic progression are associated with increasing genetic diversity at the single cell level. The success of this project will open up three important future studies: 1) Tests of whether genetic diversity in a variety of intraepithelial neoplasms is associated with progression to malignancy. 2) Tests of whether genetic diversity is associated with the evolution of resistance to cancer prevention interventions or therapy. 3) Tests of the genetic diversity of pre-clinical models of neoplastic progression so that we can develop models that faithfully recapitulate the genetic diversity of sporadic human neoplasms. Such models will be important for estimating the likelihood that resistance will develop to cancer prevention agents and provide tools for studying the management and prevention of such resistance. To address these problems we utilize expertise in caner biology, evolutionary biology and computational biology, as well as access to a tissue bank of biopsies from a Barrett's esophagus cohort that has been collected prospectively since 1989. This provides an opportunity to rapidly translate credentialed biomarkers into phase IV biomarker studies and from there into the clinic.